Semaphorin 3A (Sema3A) is implicated in repulsive axon guidance throughout the developing nervous system and during regeneration. It is necessary and sufficient for repulsion of axons by a variety of tissue explants in vitro and, in Sema3A -/mice, many of these axons improperly invade areas that normally express Sema3A mRNA. This correlation between Sema3A mRNA and robust repulsion applies to taste axons during their growth from the geniculate ganglion to the tongue, but not during pathfinding within the tongue: taste axons grow toward and then penetrate the Sema3A mRNA- rich dorsal epithelium. Clearly, there is a decrease in responsiveness to Sema3A by taste axons, or a decrease in Sema3A repellent activity. The experiments in this application are designed to determine if and when changes in repellent activity and axon responsiveness occur. The taste system is especially well suited for testing axon guidance hypotheses. Taste axons are not guided by other nerves, so explant preparations for in vitro perturbation studies do not eliminate cells involved in guidance. Also, a novel in situ organ culture method was developed in which innervation proceeds as in vivo (and can be perturbed). The response of a growth cone to a guidance cue can be reversed by other molecules in the pathway, so in situ assessment of guidance cue function is essential. Approaches. Collagen gel co-cultures will be used to determine if axon outgrowth from geniculate ganglia dissected from rat embryos at intra- lingual pathfinding stages is repelled by Sema3A-transfected cells. Whether dorsal tongue explants are repellent in vitro and whether the magnitude of the repulsion partially decreases will also be assessed. -Sema3A or mock-transfected cells will be transplanted into the tongues of organ cultured rat jaws and repulsion of taste axons in situ will be assessed. - Whether axons prematurely contact the dorsal epithelium in Sema3A -/- mice will be evaluated. Mechanisms likely to underlie the decrease in responsiveness or activity will also be tested. - If responsiveness is decreased, antibody staining will be used to determine if Sema3A receptor complex components (neuropilin-1, plexin A1 or A2, and L1) are downregulated in taste axons. - If repellent activity is decreased, Western blots will be used to determine if Sema3A is expressed at detectable levels and if it is degraded. The role of the protease furin, previously implicated in Sema3A inactivation, will be tested in vitro using a specific inhibitor.